Journal of Environmental Biotechnology
Vol. 7, No. 1, 45–53, 2007

Original paper (regular paper)

Fluorine Elimination from 4-Fluorobenzyl Alcohol by Pseudomonas spp.
TOSHINARI MAEDA, DAISUKE OKAMURA, MASAYA YOKOO, EISAKU YAMASHITA and HIROAKI I. OGAWA*
Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu
Institute of Technology, 2–4 Hibikino, Wakamatsu-ku, Kitakyushu 808–0196, Japan* TEL: +81–(0)93–695–6059 FAX: +81–(0)93–695–6012* E-mail: ogawahi@life.kyutech.ac.jp(Received; 2 October, 2006/Accepted; 15 February, 2007)
Pseudomonas aeruginosa strain IE653 can use 4-fluorobenzyl alcohol (4FBA) as a sole source of carbon and energy,
whereas P. aeruginosa strain PAO1 and P. putida strain mt-2 cannot utilize it. This strain converted 4FBA into 4-fluorobenzoic acid (4FBZ), 4-fluorophenol (4FP) and 4-fluorocatechol (4FCAT), and further into 3-fluoromuconate or fluoro-2hydroxyl-muconic semialdehyde via ortho or meta cleavage. Strain mt-2 also converted 4FBA into the same metabolites as
those detected in IE653 except 4FP. Contrary to an abundant accumulation of 4FBZ in strains PAO1 and mt-2, IE653 transiently accumulated and gradually biotransformed 4FBZ. The fluoride ions from 4FBA by P. aeruginosa strain IE653 werereleased after 8-hour cultivation, indicating that this strain was able to eﬃciently eliminate them from 4FBA metabolites
much better than strains P. aeruginosa PAO1 and P. putida mt-2 that have less eﬀective on eliminating fluoride ions. An
elimination of fluoride ions in strain IE653 was initiated after metabolism of 4FCAT, judging from the results that this strain
immediately released from 4FCAT. It was implied that ortho cleavage pathway of strain IE653 is major pathway for the
release of fluoride ions, unlike meta cleavage pathway, which had only a slight eﬀect. The eﬃcient release of fluoride ions by
P. aergunosa strain IE653 could contribute to develop the bioremediation technology for elimination of fluorine and detoxification of fluoroaromatics.
Key words: fluoroaromatics, elimination of fluorine, pseudomonad strains, metabolic pathway, ortho pathway

1.

Introduction

The advances in organic synthesis have led to the introduction of numerous new organic compounds into the environment, whose susceptibilities to biotreatment processes aremostly unknown. Fluoroaromatics are being increasingly
used in a wide range of agrochemical and pharmaceuticalproducts (herbicides and fungicides), due to the need to findenvironmentally acceptable alternatives to chlorinated compounds24,41,47). The diversity of structures and the chemical
inertness of many halogenated organic compounds pose
particular problems and challenges for microbial degradation13). Some scientists proposed that the recalcitrance of a
halogenated organic compound usually becomes greaterwith the increase of the electronegativity of the substituents;
thus, the recalcitrance of F-C is greater than that of Cl-C,
Br-C and I-C12). Also, the exceptional biological activities offluorinated aromatic compounds can be explained by thedichotomic chemical behavior of the fluorine substituent
with its +M and –I effects together with its hydrogenresembling size, reflected by the van der Waals radii of 120
pm (for H) and 135 pm (for F)35). This relationship showsthat monofluorobenzoates undergo dioxygenation, forming
fluorocatechols, or become subject to anaerobic degradation
by benzoate-degrading bacteria40). On the other hand, theexchange of a hydrogen atom by a fluorine atom can create
analogs that precede analog normal metabolic pathways
until the fluorine substituent produces a specific enzyme
inhibitor. Such a metabolic transformation is termed lethal
synthesis36). This eﬀect was found with fluoroacetate andfluorocitrate as dead-end products1,11). The critical step inthe degradation of fluorohydrocarobons is the fission of the
carbon-fluorine bond. For the cleavage of this bond in fluoroacetic acid, a rather specific enzyme has been found15,45).
In monofluorobenzoates, fluoride release can occur after
oxidative decarboxylation and ring fission of fluorocatechol16,18,42). Alternatively, the fluorine is replaced in adioxygenase-catalyzed reaction, as in the degradation of
2-fluorobenzoate, in which unsubstituted catechol and3-fluorocatechol are the products of metabolic transformation6,28,47).
The biodegradation of a vast range of halogenated aromatic compounds, especially chlorinated compounds, hasbeen described17,37), but scant information is available on themetabolic and cometabolic fate of fluorinated aromaticcompounds in bacteria. Examples of biodegradation of fluorinated compounds most commonly found in the literature
involve fluorobenzoic acids9,18,28,33,43) and fluorophenols3,4,38,40).
These literatures have reported on classic pathways, inwhich brilliant bacteria convert fluorinated compounds andMAEDA et al.

46

ultimately eliminate fluoride ions from them, and biodegradability. However, to our knowledge, relationshipbetween the release of fluoride ions and metabolic pathway
has not yet been reported. As an example, though P.
aeruginosa strain PAO1, in which genome project has finished, has probable haloacid dehalogenase in range 887583
to 888284 of genome map at the Pseudomonas Genome
Project (http://www.pseudomonas.com), this strain was notable to release the fluoride ions from fluoroaromatics(results in this study), indicating that their eﬃcient elimination may depend to metabolic pathway of fluoroaromatics.
Benzyl alcohol (BA) has been reported to form glutathione conjugates7,26) and DNA adducts48), to cause perturbations in river function and decreased clearance7,27), has demonstrated potential carcinogenicity in experimental
animals14,27). There is currently very little literature available
on toxicity of 4-fluorobenzyl chloride (4FBCl), a structural
analogue of BA. However, 4FBCl may be more risky thanBA because F-C bond is the recalcitrance12). Also, 4FBCl isspontaneously converted to 4-fluorobenzyl alcohol (4FBA)
in water although at a slower rate; however, 4FBA is still arecalcitrant compound, and forms N-acetylcysteinyl conjugates2). We screened 4FBA-biodegradaing bacteria fromindustrial eﬄuents in Kitakyushu, Japan, and found 1
strain. One strain was designated as P. aeruginosa strain
IE653, judging from the results of characterization with
NF-18 and API20NE systems, and homology search of 16S
ribosomal RNA gene. This paper describes that biotransformation and detoxification of 4FBA by P. aeruginosa strain
IE653 were much higher than those by strains P. aeruginosastrain PAO1 and P. putida strain mt-2 (a toluene degrader),and that strain IE653 could eﬃciently eliminate the fluoride
ions from fluoroaromatics.

2.
2.1.

Materials and Methods

Chemicals

We used 4-fluorobenzyl alcohol (4FBA) (Merck KGaA,
Germany), 4-fluorobenzoic acid (4FBZ), 4-fluorophenol(4FP), 4-fluorocatechol (4FCAT) (Tokyo Chemical Industry
Co. Ltd., Japan), benzyl alcohol (BA), catechol (CAT)(Wako Pure Chemical Industries Ltd., Japan), benzoic acid
(BZ) and cis,cis-muconic acid (Kanto Kagaku Co. Ltd.,
Japan) as chemical standards. All chemicals were of thehighest purity commercially available.
2.2.

Bacterial strains and culture conditions

Bacteria used in this study were P. aeruginosa strains
IE653 and PAO1, and P. putida strain mt-2. These bacteria
were aerobically grown until the late logarithmic growthphase in M9 minimal medium41) containing benzyl alcohol(1 mM) and succinic acid (1 mM) as carbon source at 30°C
in the dark with shaking (120 rpm). Cells were washed twicewith 0.1 M sterilized phosphate buﬀer (pH 7.5) and resuspended in the same buﬀer. Cell suspensions were mixed into
reaction solution, and then the mixture was aerobically

incubated at 30°C in the dark with shaking (120 rpm).
2.3.

Determination of viable cells

M9 minimal medium (100 mL) with and without a carbon source (1 mM succinic acid or 1 mM 4FBA) was mixed
with cell suspensions (a final concentration; about 1×
106 cells/mL), and then the mixture was aerobically incubated at 30°C in the dark with shaking. Periodically, serial
dilutions of the reaction solution were spread on LuriaBertani (LB) agar plate (10 g tryptone, 5 g yeast extract, 5 g
sodium chloride and 15 g agar/L), and then these plates
were incubated at 30°C for 18 h. We counted the colonies
to calculate colony forming units (cfu).
2.4.

Identification of 4FBA metabolites

The cells (about 5×108 cells/mL) were aerobically grownfor diﬀerent periods at 30°C and were removed by centrifugation at 5000×g for 10 min. The culture supernatant(adjustment of pH with 6 N HCl; pH 3.0) was extracted
3 times with 100 mL ethyl ether, then the extracts weredried over anhydrous sodium sulfate, and excess solvent wasremoved by rotary evaporation at 30°C. When required, thederivatization of samples (trimethyl silylation or methylation) was carried out. Gas chromatography-mass spectrometry (GC-MS) analyses of these samples were performed
with a HP6890 Series GC system/5973 Mass Selective
Detector (Hewlett Packard, USA). 4FBA metabolites wereidentified by comparison of retention times and mass
spectra between samples and standard chemicals. Also, themetabolites (after complete elimination of excess solvent)were dissolved in 600 µL of 0.1 M phosphate buffer(pH 7.0). Then samples were transferred to NMR tubes,
and D2O (100 µL) was added to provide a lock signal. The
mixtures were analyzed by 19F nuclear magnetic resonancespectroscopy (19F NMR) with a Burker Avance 400S
(400 MHz) spectrometer (Germany).
2.5. Measurement of 4FBA, its metabolites
and fluoride ion
4FBA and its metabolites were determined by highperformance liquid chromatography (HPLC). HPLC analyses were performed on an Inertsil ODS-2 column (GLSciences Inc., Japan) with acetonitrile-water (25 : 75) as themobile phase, with a flow rate of 0.4 mL/min. All compounds were detected at 254 nm with a Shimadzu SPD10AVP UV-VIS detector (Japan). Fluoride ion concentrations were measured with a TOA Ion Meter IM-40S (Japan)
containing a fluoride-selective lanthanum fluoride electrodeas previously described34). Concentrations of 2-hyroxymuconic semialdehyde and fluoro-2-hydroxymuconic semialdehyde were calculated from the molar absorptivity25,31).
2.6.

Preparation of extracts

The cells (P. aeruginosa IE653) were grown at 30°C withshaking for 18 h in 4FBY medium (M9 with 0.2% 4FBA
and 0.05% yeast extract). Cultures were harvested by

Detoxification of fluorine aromatics by pseudomonad strains

centrifugation (5000×g, 10 min), and the cells were washedtwice with 0.1 M phosphate buﬀer (pH 7.5). Cells weresuspended in this buﬀer at a density of 7×109 to 1010 cells/
mL and disintegrated for 5 min in an ice bath with a Branson ultrasonics, model Sonifier 450 (Central Scientific Commerce Co. Ltd., Japan). The preparation was centrifuged at
15000×g for 20 min at 0°C to remove whole cells and large
debris. Protein was measured by the method of Bradford
using bovine serum albumin as a standard5).
2.7.

4FCAT biotransformation by cell extracts.

Enzyme assays of catechol-1,2-dioxygenase and catechol2,3-dioxygenase were performed at 30°C with a UV-VIS
Spectrophotometer V-530 (JASCO, Japan). Catechol-1,2dioxygenase and catechol-2,3-dioxygenase activities were assayed by measuring the formation of cis,cis-muconate and2-hydoxymuconic semialdehyde, respectively. The molar
absorption coeﬃcient was 16800 at 260 nm for cis,cismuconate25) and 42000 M–1 cm–1 at 375 nm for 2-hydroxymuconic semialdehyde8). One unit of these enzyme activities
was defined as the formation of 1 µmol of the product permin. Specific activity was calculated as the formation of
1 µmol of the product/min/mg-protein. All measurements
were made in cuvettes with a 1-cm light path in a total volume of 3.0 mL.
The enzyme activity of catechol-2,3-dioxygenase is inactivated by H2O230). We used cell extracts to research which
pathways (ortho and meta cleavage pathways) are essential
for the elimination of fluorine from 4FCAT in biotransformation. Catechol-2,3-dioxygenase in cell extracts wasinactivated as follows. H2O2 at a final concentration of
80 mM was added into the extracts (6.8 mg-protein/mL)
and incubated at 30°C for 30 min. Then bovine liver catalase
was added (100 enzyme units/mL) followed by incubatedfor another 10 min to destroy excess H2O2.
The enzyme activities in H2O2-treated or untreated extracts
were assayed in 200 mL of 0.1 M phosphate buﬀer (pH 7.5)
containing 0.33 mM 4FCAT. Fluoride ion concentrations
were measured to determine a fluorine-releasing activity.

3.
3.1.

47

Results

Growth curve of bacterial strains in various conditions

We isolated P. aeruginosa strain IE653, which was able
to biodegrade 4FBA, and tested whether this strain canutilize it as a sole carbon source. Two strains, P. aeruginosa
PAO1 that is a delegate among various pseudomonadstrains and P. putida mt-2 known as a toluene degrader,were used as control strains in this study. We used these twostrains because the whole genome sequences of these bacteria have been completely determined and the metabolic
pathway by P. putida mt-2 has been intensively investigated. Strain IE653 grew in the medium supplemented with
4FBA as a sole carbon source, whereas PAO1 could not
grow and strain mt-2 showed only slight growth (Fig. 1).
These results indicate that strain IE653 can utilize 4FBA asa sole carbon source.
3.2.

4FBA metabolism pathways by pseudomonad strains

GC-MS analyses identified 4-fluorobenzoic acid (4FBZ)
as a major metabolite, and simultaneously 4-fluorophenol(4FP), 4-fluorocatechol (4FCAT) and fluoro-2-hydroxymuconic semialdehyde (FHMS) as minor products in theculture supernatant of strain IE653 (Fig. 2). In addition,
19
F-NMR analyses demonstrated that P. aeruginosa IE653
produced three metabolites (4FBZ, 4FP and 4FCAT [Fig.
3-A]) and 3-fluoromuconate (3FM) as shown in Fig. 3-B.
On the other hand, P. putida mt-2 converted 4FBA into
the same metabolites except 4FP as those detected in theculture supernatant of strain IE653. Enzyme assays using
cell extracts of IE653 showed that this strain has two ringcleavage pathways, in which catechol and 4FCAT werebroken down by catechol 1,2-dioxygenase and catechol
2,3-dioxygenase, producing cis,cis-muconate and 3FM, and2-hydroxyl muconate semialdehyde and FHMS, respectively (Table 1); these results are consist with the structural
results using GC-MS and 19F-NMR. On the other hand, we
implied that there is the possibility that IE653 produces
4-fluoro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid

Fig. 1. Growth curves of pseudomonad strains (A, P. aeruginosa strain IE653; B, P. aeruginosa strain PAO1; C, P. putida strain mt-2) in M9with 1 mM succinic acid (●) and 1 mM 4-fluorobenzyl alcohol (▲), and without a carbon source (×).

48

MAEDA et al.

Fig. 2. GC-MS spectra of 4-fluorobenzyl alcohol metabolites produced by P. aeruginosa IE653. (A) 4-fluorobenzoic acid, (B) 4-fluorophenol,(C) 4-fluorocatechol, and (D) fluoro-2-hydroxyl-muconic semialdehyde.

Fig. 3. 19F-NMR spectra of 4-fluorobenzyl alcohol metabolites produced by P. aeruginosa IE653. (A) Metabolites from 4-fluorobenzyl alcohol; arrows indicate 4-fluorobenzoic acid, 4-fluorophenol and 4-fluorocatechol. (B) Metabolite from 4-fluorocatechol; arrow indicates
3-fluoromuconate.

(4FCHDC), which is a intermediate product during conversion from 4FBZ to 4FCAT, judging from the comparison ofmass fragment patterns in mass spectra between 4-chloro3,5-cyclohexadiene-1,2-diol-1-carboxylic acid and 4FCHDC(it is possible to analyze the mass fragment patterns in chlorinated compounds because Cl has stable isotopes (35Cl and37
Cl; 3 : 1 of ratio)) as shown in Fig. 4. This result implies
that P. aeruginosa IE653 may have two pathways (4FBZ→
4FCHDC→4FCAT and 4FBZ→4FCHDC→4FP→4FCAT)
whereas strain mt-2 has a single pathway (just 4FBZ→
4FCHDC→4FCAT) in metabolism from 4FBZ to 4FCAT.
3.3.

4FBA biotransformation and 4FBZ accumulation

Figure 5 indicates the patterns of 4FBA biotransformation and 4FBZ accumulation in three pseudomonad strains.

Strain mt-2 biotransformed 4FBA much better than strains
IE653 and PAO1. Strains IE653 and PAO1 showed thesame level of biodegradation rate. When P. aeruginosa
IE653 was cultivated with 4FBA, 4FBZ was transiently
accumulated and was then gradually decreasing with time.
In contrast, strains PAO1 and mt-2 accumulated a highconcentration of 4FBZ. However, they did not greatly
accumulate BZ when cultivated with BA (Table 2). These
results indicate that fluoride substitution in benzoic acidleads to an increased resistance to biodegradation by strains
PAO1 and mt-2, while strain IE653 eﬃciently transforms
fluoroaromatics. Also, the accumulations of meta cleavage
products (yellow products), FHMS and HMS, which havean absorption maximum at 382 and 375 nm, respectively,
were observed in strains IE653 and mt-2 (Table 2).

Detoxification of fluorine aromatics by pseudomonad strains

49

Fig. 4. Production of putative intermediates, 4-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (A) and 4-fluoro-3,5-cyclohexadiene-1,2diol-1-carboxylic acid (B) by P. aeruginosa IE653. These mass spectra were obtained by GC-MS analyses.

Table 1. Enzyme activity for catechol 1,2-dioxygenase and catechol 2,3-dioxygenase and fluorine elimination by
crude extracts of strain IE653.
Extracts
untreated
H2O2-treatedFluoride ion concentration (mg/L)
1h
3h7h
0.7±0.1
1.8±0.2

1.41±0.05
2.7±0.1

2.0±0.2
2.75±0.05

Specific activity (units/mg protein)aorthobmetac
0.23±0.05d
0.32±0.02

0.05±0.02
0.001±0.000

Values are the means ± the standard deviations of two independent measurements.
For definition of specific enzyme activity see Materials and Methods.
bcatechol 1,2-dioxygenaseccatechol 2,3-dioxygenased
Initial velocity was determined, since the reaction reached the plateau in approximately 15 sec.
a3.4.

Fluorine elimination from 4FBA metabolites
by pseudomonad strains

Complete detoxification of fluoroaromatics will require
an elimination of fluoride ion from such compounds. We
tested whether P. aeruginosa strains IE653 and PAO1, andP. putida strain mt-2 can release the fluoride ions from
4FBA or its metabolites to reveal a relationship between
metabolic pathways and fluorine elimination. The highaccumulation of fluoride ion was observed in the reaction
mixture containing strain IE653 after 8-hour cultivation(Fig. 6-A), although biodegradation of 4FBA was initiatedby IE653 within 8 h (Table 2 and Fig. 5). These results indicate that the fluorine is eliminated from 4FBA metabolites
not 4FBA itself. Other two strains were only slightly eﬀective for fluorine elimination. On the other hand, fluoride
ions from 4FCAT were immediately detected in strain IE653(Fig. 6-B), indicating that this strain eliminates them after
the conversion to 4FCAT. Strains PAO1 and mt-2 released
fluoride ions at a much slower rate. P. aeruginosa IE653
could not eliminate the fluoride ions from fluoroacetate(data not shown).

MAEDA et al.

50

Table 2. Mass balance of 4FBA metabolites and BA metabolites by pseudomonad strains.
IE653
Time
(hour)
4
8
24

4FBA biodegradation
Concentration of chemicalsa (µM)

BA biodegradation
Concentration of chemicals (µM)

4FBA

4FBZ

FMS

FHMS

BA

BZ

MS

HMS

580±4b
410±7
211±4

260±5
122±8
81±1

NDetermc
NDeterm
NDeterm

15±3
54±5
19±3

712±9
365±5
104±1

120±2
60±8
75±2

40±5
26±2
15±3

20±5
15±3
7±1

mt-2
Time
(hour)
4
8
24

4FBA biodegradation
Concentration of chemicals (µM)

BA biodegradation
Concentration of chemicals (µM)

4FBA

4FBZ

FMS

FHMS

BA

BZ

MS

HMS

450±7
101±7
NDetectd

221±3
320±2
561±6

NDeterm
NDeterm
NDeterm

28±5
75±2
27±2

420±3
50±2
NDetect

220±2
151±9
72±4

21±5
9±7
6±1

21±1
11±1
7±3

PAO1
Time

4FBA biodegradation
Concentration of chemicals (µM)

BA biodegradation
Concentration of chemicals (µM)(hour)
4

4FBA

4FBZ

FMS

FHMS

BA

BZ

MS

HMS

611±6

289±2

NDeterm

NAe

8
24

432±3
232±5

519±2
430±5

NDeterm
NDeterm

NA
NA

550±6
410±2
202±1

292±2
271±3
150±3

NDeterm
NDeterm
NDeterm

NA
NA
NA

4FBA, 4-fluorobenzyl alcohol. 4FBZ, 4-fluorobenzoic acid. FMS, fluoromuconate. FHMS, fluoro-2-hydroxymuconic
semialdehyde. BA, benzyl alcohol. BZ, benzoic acid. MS, muconic acid. HMS, 2-hydroxymuconic semialdehyde.
bValues are given ± standard deviations.
c
Not determined.
d
Not detected.
e
Not applicable.
a3.5. Fluorine elimination pathway
P. aeruginosa strain IE653 has ortho and meta cleavage
enzymes, which cleavage carbon-carbon bonds of catechol
in position of C-1,2 and C-2,3, respectively (Table 1). We
tested whether the fluorine-elimination pathway is ortho
pathway or meta pathway, or both. The active form of ironin catechol 2,3-dioxygenase is in a ferrous state and the
enzyme activity is inactivated by H2O2 due to the conversionof the active ferrous form to the inactive ferric form32). In
contrast, the active form of iron in catechol 1,2-dioxygenase
is a ferric iron and thus resistance to the oxidant29). Therefore, we used this phenomenon for examining an elimination of fluorine from fluorinated compounds. The activityof catechol 2,3-dioxygenase in crude extracts of strain IE653
was completely inactivated by H2O2 although catechol
1,2-dioxygenase was active after H2O2 treatment (Table 1).
Furthermore, the inactivation of meta cleavage enzyme
resulted in an increased activity of ortho cleavage enzyme.
It is probably due to increased amonts of substrate (catechol)available for catechol 1,2-dioxygenase because of inactivation of the competitor, catechol 2,3-dioxygenase. The
fluoride ions were detected from the solution with untreatedand H2O2-treated cell extracts (Table 1). However, a higher
concentration of fluoride ion was produced in the reaction
mixture with H2O2-treated crude extracts than that withuntreated extracts. These results suggest that ortho pathway
may be a major pathway for fluorine elimination.

4. Discussion
The utilization of 4-fluorobenzoate as a sole source ofcarbon and energy has been described earlier for several
bacterial genera including Pseudomonas18,33,42,44,46), Paracoccus23), Acinetobacter49), Corynebacterium21,22), Alcaligenes33) and Aureobacterium33). We isolated P. aeruginosastrain IE653, which can utilize 4FBA as a sole energy(Fig. 1), from industrial eﬄuents in Kitakyushu, Japan.
This strain IE653 converted 4FBA to 4FBZ, 4FP, 4FCAT,
3FM and FHMS. 19F NMR spectra of 3FM (for chemical
shift [–108 ppm] and coupling constant [22 and 32 Hz] wereconsistent with the results of Boersma et al.3). Also, P. putida
mt-2 produced the same metabolites as strain IE653 but not
4FP, in agreement with the results of the previous studies19,20).
BZ or 4FBZ oxidation pathway described in this paper
resembles the toluene dioxygenase pathway. In the toluene
dioxygenase pathway, toluene is oxidized to toluene cis
glycol, which is further oxidized to 3-methylcatechol. It is
known that the first intermediate, toluene cis glycol, is nonenzymatically dehydrated to cresol10,39). Therefore, it ispossible that 4FP is produced from 4FCDHC through nonenzymatic or enzymatic dehydration and decarboxylation.

Detoxification of fluorine aromatics by pseudomonad strains

51

Fig. 6. Release of fluoride ions from 4FBA (A) or 4FCAT (B) by P.
aeruginosa strains IE653 (○) and PAO1 (●), and P. putida
strain mt-2 (▲).
Fig. 5. Biotransformation of 4FBA by P. aeruginosa strains IE653(○) and PAO1 (●), and P. putida strain mt-2 (▲). (A) biodegradation of 4FBA. (B) accumulation of 4FBZ.

Further investigation is required to elucidate eﬀects of 4FP
formation on eﬃciency of 4FBZ degradation in P. aeruginosa IE653.
4FBZ, which is a major product of 4FBA, was resistanceto degradation by P. aeruginosa PAO1 and P. putida mt-2
whereas P. aeruginosa IE653 was able to gradually biodegrade 4FBZ. Interestingly, strain IE653 accumulated a highconcentration of 4-chlorobenzoate (4CBZ) when it was incubated with 4-chlorobenzyl alcohol (data not shown).
These results suggest that enzymes involved in 4FBZ oxidation in IE653 cannot oxidize 4CBZ. The metabolisms of
4FBZ, 4CBZ and BZ may be key steps for eﬃcient biotransformation of cognatic aromatics. Strains PAO1 andmt-2 accumulated 4FBZ from 4FBA but not BZ from BA,
suggesting that fluorine substitution in benzoate hampers
further degradation of 4FBZ in strains PAO1 and mt-2. We
will need to reveal why P. aeruginosa IE653 can biodegrade
4FBZ eﬀectively and whether the second pathway (producing 4FP) in IE653 is important for this insight, and investigate the diﬀerence of structure or specificity of enzymes involved in degrading 4FBZ.
Results in Fig. 6 suggest that fluorine is eliminated from
4FCAT or its metabolites but not from 4FBA. However, we
do not know which compound(s) is a substrate of fluorine
elimination. Schreiber et al.44) reported that 3FM produced

by ortho cleavage of 4FCAT was further converted to maleylacetate via cis-dienelactone. They suggested that a nonenzymatic fluorine elimination from 4-fluoromuconolactone
occurred in the reaction. Further investigations are required
to eludidate whether removal of fluorine from metabolites
of 4FBA by P. aeruginosa IE653 is enzymatic or chemical
reaction.
P. aeruginosa strain IE653 can eﬃciently biotransform
4FBA and can detoxify it via an elimination of fluorine
from it. Our strain may be valuable to develop the bioremediation technology for elimination of fluorine and detoxification of fluoroaromatics.

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